Background
Breast cancer is the most commonly diagnosed invasive cancer in females [
1] and is most often an estrogen (17β-estradiol) driven tumour [
2,
3]. The primary cellular mediator of estrogen is the intracellular transcription factor estrogen receptor alpha (ERα), which is expressed in 75 % of early breast cancers [
4]. ERα and PR positivity as assessed via immunohistochemistry of primary breast cancer is currently the gold standard indicator for hormonal therapy, applied either at the time of diagnosis or subsequent to surgical, chemotherapeutic and/or radiation management. While the molecular mechanisms and consequences of estrogen-mediated action have received considerable research attention, the molecular mechanisms of progesterone signalling have not been as widely reported. More recently PR is emerging as a key mediator of normal mammary gland development and tumorigenesis in mice, promoting mammary stem cell expansion and directing the immune microenvironment [
5‐
10].
The majority of the cellular effects of progesterone are mediated by the progesterone receptor (PR), an intracellular transcription factor of which two isoforms exist, PR-A and PR-B. Because
PR is an estrogen regulated gene, the expression of PR protein detected by immunohistochemistry as a diagnostic tool was found to discriminate between those most likely to respond to endocrine therapy, from those that will not [
11,
12]. Indeed, expression of PR in breast cancer in the absence of ERα is rare (1.5 % of cases), and evidence suggests that such cases may represent false negatives for ERα staining upon re-analysis [
13‐
16]. Nevertheless, PR appears to be more than a mere diagnostic indicator of estrogenic activity, as clinical studies have demonstrated it to be an independent biomarker of endocrine therapy response as well as a prognostic biomarker in postmenopausal breast cancers [
12,
16‐
18]. Smaller studies in premenopausal women have found that tumours containing higher PR positivity had the best response to tamoxifen [
19].
In premenopausal women, the physiological role of progesterone is inextricably linked to that of estrogen, with regards to production and secretion by the ovaries during the menstrual cycle. Increased production of estrogen by the maturing follicles ultimately results in ovulation, after which the corpus luteum produces and secretes progesterone. The secretion of progesterone in turn acts on the adrenal glands to stimulate a concomitant secondary, albeit smaller, peak of serum estrogen [
20]. Evidence also suggests that the postmenopausal breast is capable of sequestering and/or synthesising progesterone and estrogen from circulating hormonal precursors [
21‐
25]. Collectively, it appears most likely that PR is activated within a hormonal milieu that includes active estrogen signalling.
Genomic and functional studies of receptor action
in vitro now provide unprecedented detail into the precise mechanics of ERα and, to a lesser extent, PR action in breast cancer cells. Those for PR have, however, been exclusively performed in the absence of exogenous estrogen [
26‐
31]. Binding of estrogen by ERα and progesterone by PR results in association of the receptors with specific sites on chromatin. Receptor binding to DNA subsequently directs the recruitment of cofactors and associated coactivators and corepressors, resulting in modification of the local chromatin landscape and activation or repression of target genes. Indirect tethering of the receptors to chromatin has also been observed via interaction with DNA-bound factors such as AP-1, Stat3 and SP1 [
27,
32,
33]. Despite the findings that PR expression is almost always accompanied by ERα expression [
16], to date there are few reported studies investigating progesterone transcriptional signalling and PR binding in the context of estrogen-mediated signalling. Indeed, most studies of PR DNA binding have been performed in T-47D breast cancer cells that do not depend upon estrogen for PR expression [
34]. In this report, we demonstrate a 10-fold induction in PR binding upon progesterone treatment in estrogen pre-treated versus non estrogen treated ZR-75-1 cells and demonstrate that progesterone and estrogen cotreatment drive a unique gene expression profile in ZR-75-1 that is distinct from treatment with either hormone alone, which includes up-regulation of signalling mediators of ErbB pathways. Estrogen and progesterone cotreatment cause significant changes to the predicted intrinsic breast cancer subtype, specifically to one that resembles more aggressive, therapy resistant disease.
Discussion
In a recent meta-analysis, breast tumour subtyping via the Oncotype DX® platform was found to guide clinical decision making regarding the use of adjuvant chemotherapy in 34 % of early breast cancer cases [
63,
64]. Moreover, the St Gallen International Expert Consensus found that microarray-based intrinsic subtype classification of breast cancers is an important guide for chemotherapy use in ERα positive, HER-2 negative disease [
65]. That panel did however recognise the potential prohibitive cost of wide-spread multigene expression analysis, and instead propositioned immunohistochemical surrogates such as dichotomising ERα positive breast cancer cases on the basis of PR and Ki67 positivity thresholds and HER2 status, even though such measures have been found to be less accurate [
17,
60,
65]. Despite increased recognition and utility of subtype classification in the clinical setting, the factors or conditions that drive individual tumours into classifiable subtypes are currently unknown. Even though this study was conducted in breast cancer cell lines, the findings of this study suggest that exposure to hormones may alter the transcript profile of breast cancer cells sufficiently to change their classification by multi-gene algorithms. Specifically, we found that estrogen pretreated breast cancer cells exhibit a Luminal A subtype, which switches to a Basal-like subtype upon combined estrogen and progesterone treatment. In support of steroid-induced effects on intrinsic subtypes, the incidence of Basal-like tumours decreases significantly with age, from 44 % in premenopausal aged patients (21–39 years) to just 9 % in patients aged 70–93, who exhibit lower, more static serum levels of progesterone and estrogen [
66]. Indeed, the expression of PR and other key estrogen regulated genes in breast tissue from postmenopausal women is positively associated with serum estrogen levels [
67]. In the pre-menopausal setting, a study of estrogen regulated genes throughout the menstrual cycle in early breast cancer samples demonstrated a significant increase in PR transcript and protein levels during follicular and luteal phases (days 7–26), corresponding with higher known circulating estrogens [
68]. Likewise, the expression of PR, a PR regulated gene
RANKL, and an ERα regulated gene,
TFF1 are all significantly higher in premenopausal in comparison to postmenopausal women [
69]. Recent studies demonstrate intra-individual variability in multigene signature scores between fine needle biopsies and resection specimens [
70]. Finally, PR abundance may decrease upon activation by progesterone treatment, adding to the complexity of using PR abundance as a surrogate for intrinsic subtype status [
57]. While the study reported herein is provocative, these findings require careful validation in premenopausal breast cancer patients. In the meantime, these data suggest that careful consideration be given to the menopausal status of women, and the concentration of circulating estrogen and progesterone at the time of tumour collection, if RNA-based subtyping tools, and perhaps their immunohistochemical surrogates are to be used in clinical decision making.
The potential for plasticity between the intrinsic subtypes of breast cancer has not been widely investigated. From a clinicopathological perspective, nearly 70 % of Basal-like tumours and just 3 % of Luminal A tumours have a triple negative phenotype (ERα and PR negative and no HER2/neu overexpression) [
71], and 65 % of ERα negative/PR positive tumours exhibit a Basal-like PAM50 subtype [
63‐
65]. Furthermore, tumours arising in younger women have significantly lower ERα and PR expression, but higher HER-2 and EGFR expression [
72], and in Basal-like breast cancers and breast tumours in younger women, the level and expression of EGFR is an adverse prognostic factor [
72,
73]. While the studies contained herein are preclinical in nature, we describe that combinatorial estrogen and progesterone treatment result in upregulation of several key members of the EGFR signalling pathway. If this relationship is verified in premenopausal and postmenopausal breast cancers, it is possible that subtyping tools developed predominantly from postmenopausal women may be particularly prone to menstrual cycle-induced plasticity or hormone-driven artefacts.
In ERα positive breast cancers, PR positivity is indicative of a more favourable response to endocrine therapy [
16], but does not distinguish between a clinical response to tamoxifen or aromatase inhibitors [
18,
74]. Nonetheless, the percent and intensity of breast cancer cells positive for PR protein by immunohistochemistry is positively correlated with time to recurrence in both tamoxifen and anastrozole treated patients, and Luminal A type breast cancers containing more than 20 % PR positive cells have a better prognosis than those with less than 20 %, independent of endocrine therapy [
59]. Thus, while abundance of PR provides prognostic information beyond ERα positivity, the important question is whether this derives from the intrinsic biological activity of PR, or is purely due to PR acting as a marker of the extent of tumour cell ERα activity or responsiveness. The intrinsic biological role of PR has been difficult to study in breast cancers precisely because of its dependent relationship on ERα, and the concordance between levels of ERα and PR in breast cancers [
12,
34,
75]. We show here that PR action is dependent on the hormonal context, with concurrent estrogen treatment producing a unique transcriptomic response to progesterone. Combined with our finding that the master regulator of a progesterone response in breast cancer cells appears to be estrogen, which regulates PR abundance, thereby permitting PR DNA binding, our findings suggest that the actions of estrogen and progesterone are inextricably linked. Interestingly, the ancestral vertebrate steroid receptor was a receptor that preferentially bound estrogens, with the progesterone receptor the second steroid receptor to evolve [
76,
77]. Hence, the estrogen and progesterone receptors have the longest coexistence in relation to the other steroid receptors, so perhaps it is not surprising that a complex functional regulatory relationship exists between them, where ERα-mediated upregulation of PR abundance permits activity in response to progesterone, and PR in turn, regulates a subset of ERα actions [
78,
79]. Mechanistically, we anticipate that a large part of the unique response observed here is the sensitization to progesterone mediated by upregulation of PR by estrogen, resulting in a combined estrogenic/progestogenic response. Given that we observed a large overlap in binding sites between ZR-75-1 cells cotreated with estrogen and progesterone and those previously reported in T-47D cells treated solely with progesterone, alternative binding of the PR induced by estrogen treatment is unlikely to be the sole cause of the unique estrogen and progesterone transcriptome observed here. One possibility is that estrogen treatment may cause differential regulation of transcriptional collaborators, such as FOXA1. While further studies will determine the precise mechanism, we propose that the counter-regulation of approximately one quarter of estrogen responsive genes upon progesterone treatment, and upregulation of growth factor receptor pathways, may together contribute to the unique transcriptome observed here.
HER2 and/or EGFR overexpression is a cause of endocrine resistance, and ER positivity has been shown to decrease the effectiveness of HER2 targeting agents and provide a potential avenue for resistance to HER2-targeted therapies [
55,
80‐
86]. Many molecular and clinical studies suggest that HER2 and hormone receptor positive breast cancers have the ability to switch between hormonal-driven and ErbB-driven signalling, with this switch mediating therapeutic resistance. This suggests that each of these two pathways are sufficient to propagate cancer cell growth, with the mechanistic switch perhaps partly being explained in terms of estrogen-ERα complexes or tamoxifen-ERα complexes repressing HER2 transcription [
55]. Here, our data suggest that PR may collaborate in the relationship or interplay between hormonal and ErbB signalling. While only in a single breast cancer cell line, we demonstrate the potential for progesterone to activate EGFR signalling, consistent with progesterone potentiation of EGF responses in ZR-75-1 cells [
48]. In early breast cancers moreover, those carrying a gene signature representing activity of hyperphosphorylated PR were found to have higher prevalence of HER2 positivity and distal metastasis [
60]. Together, these findings firmly position PR as much more than a marker of ERα action in breast cancer, and our observations that both estrogen and progesterone play a role in the upregulation of growth factor receptor pathways suggest that PR targeting should be considered more closely as a partner in currently employed endocrine and ErbB-targeted therapies.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
EFN was involved in the conception and participated in the design of the study, performed the ChIP sequencing and transcriptomic studies, participated in the data analysis and drafted and revised the manuscript. LAS participated in analysing and interpreting the ChIP sequencing and transcriptomic data, critically revised the manuscript, and contributed intellectually to the study as a whole. APT was involved in the design of the study, performed transcriptomic studies, critical interpretation of data, figure construction, assisted with drafting the manuscript and provided critical review of the manuscript. LG performed the flow cytometry studies and provided important intellectual revisions to the manuscript. MAL was involved in the study conception, design and manuscript drafting. ES contributed to the design, analysis and interpretation of the data and was involved in drafting and critically revising the manuscript. PGG advised on experimental design and analysis and was involved in drafting and critical revision of the manuscript. WI revised the manuscript critically for important intellectual content and was involved in overall study design. JDG was involved in the conception, design and analysis of data and revised the manuscript critically for important intellectual content. GB co-directed the study with EFN and was involved in the conception, design, acquisition, analysis and interpretation of data and critical revision of the manuscript. All of the authors have read and approve of the final version of this manuscript.